Recent acts of bioterrorism have underscored the importance of understanding the molecular mechanisms of bacterial pathogenesis. Yersinia pestis, the causative agent of the plague or the Black Death, has been used previously as a biological weapon. There is also concern that genetic engineering has been used to generate antibiotic resistant strains of Yersinia. This proposal focuses on understanding the biochemistry of the Yersinia virulence factor known as YopT. Our laboratory has recently demonstrated that YopT is a cysteine protease. We propose to develop a highly efficient and sensitive assay for YopT's catalytic activity. We will also produce and purify recombinant YopT, a YopT/SycT complex, and a catalytically inactive YopT complexed with substrate. The purified protein(s) will be used to obtain crystalline protein suitable for structural analyses using X-ray diffraction. We have chosen the R21 venue for funding because, although we have made good progress on determining the function of this virulence protein, we have no preliminary data about its catalytic properties. In addition, even though we have determined the structure of a YopT family member, AvrPphB, it will be essential to have a structure of YopT in order to identify potential inhibitors directed against this protease.